CN108611341A

CN108611341A - The hemagglutinase and its preparation method and application extracted from spearhead viper venom

Info

Publication number: CN108611341A
Application number: CN201611131105.6A
Authority: CN
Inventors: 崔亮亮; 石皎; 李秀琳; 李秀娜; 丁忠福; 葛琳; 孙东; 薛百忠; 王宏英; 薛雁
Original assignee: Liaoning Yuanda Nuokang Bio-Pharmaceuticals Co Ltd
Current assignee: Liaoning Yuanda Nuokang Bio-Pharmaceuticals Co Ltd
Priority date: 2016-12-09
Filing date: 2016-12-09
Publication date: 2018-10-02

Abstract

The present invention provides the hemagglutinasees that one kind is extracted from spearhead viper venom (Bothrops atrox), and the present invention also provides the preparation methods of the hemagglutinase, the described method comprises the following steps：After spearhead viper venom is pre-processed, pass through 25 ion-exchange chromatographies of diethyllaminoethyl sephadex A successively；Benzamidine sepharose gel 4FF (HS) affinity chromatography；And 75 gel permeation chromatographies of sephadex G；Obtain spearhead viper venom hemagglutinase.The present invention also provides a kind of application of spearhead viper venom hemagglutinase in preparing the drug for treating hemorrhagic conditions.Compared with prior art, step of preparation process of the invention is few, and the period is short, reduces process environment exposure；Product yield is high, and technique removes that impurity ability is strong, can obtain 35% or more yield, 98% or more purity spearhead haemocoagulase；Technology stability is high, and product quality consistency is good, is conducive to clinical application safety.

Description

The hemagglutinase and its preparation method and application extracted from spearhead viper venom

Technical field

The invention belongs to biomedicine fields, and in particular to a kind of to extract hemagglutinase from spearhead viper venom, the present invention is also It is related to extracting the method for hemagglutinase and the purposes of the hemagglutinase from spearhead viper venom.

Background technology

Blood coagulation is the physiology course of complexity, is broadly divided into three phases：First stage, Stuart factor (coagulation Factor X, FX) activation；Second stage, PROTHROMBIN ACTIVATOR；Phase III, fibrinogen become fibrin.

Hemostatic is extracted from snake venom to have a long history.Currently, clinical application more successfully has Switzerland to produce Hemostatic Reptilase, i.e. Reptilase, its main component are from Brazilian snake (Bothrops Jrarace, Lachesis Atrox the snake venom thrombin-like enzyme (Snake Venom Thrombin-like Enzyme, SVTLE) extracted in venom), the enzyme Belong to the serine protease of trypsase family, there is arginine ester hydrolysing enzyme and lactamase activity.The enzyme in vitro can be by fiber Proteinogen is hydrolyzed to fibrin, without the participation of other coagulation factors, does not activate fibrin stabilizing factor in vivo, is formed Fibrin clot it is unstable, be easy to be dissolved by fibrinolytic system, to not influence the activation and release of blood platelet, do not have blood Bolt forms danger.Since batroxobin is structurally and functionally similar to human thrombin, therefore it is known as " batroxobin ".It is so far Only, batroxobin is found in more than 30 kinds of snake venom, and is successfully separated and has been purified more than 20 and plant, wherein more than 10 kind batroxobins All or part of amino acid sequence is set forth (Zheng Ying, Shen Juren, Zhang Fuqiang etc., Agkistrodon acutus hemocoagulase atrox N-terminal sequence measurement And its styptic activity analysis [J] Chinese Medical Sciences University journal).

Since the batroxobin overwhelming majority extracted in natural snake venom is acidic protein, anion exchange is mostly used Column is detached, and the purifying of the methods of attached gel filtering, affinity chromatography and reversed-phase liquid chromatography.

In the document reported, the method for extraction and purification of snake venom thrombin-like enzyme includes：

1) after pre-processing snake venom, it is cloudy to carry out diethyllaminoethyl-sephadex A50 (DEAE-Sephadex A50) 100 chromatography of ion-exchange chromatography and molecular sieve Sephadex G.The technique preprocessing process is loaded down with trivial details, cause enzymatic activity at Part loss is more, influences the Rate activity and yield of product, and it is organic molten to use in pretreatment phenol and its derivatives, methanol etc. Agent influences the purifying in later stage, is unsuitable for large-scale production.

2) after pre-processing snake venom, heparin-cyanogen bromide-Ago-Gel 4B (Heparin-CNBr-Sepharose are carried out 4B) affinity protein purification.Though method is slightly simple earlier above for the technique preprocess method, the derivative of phenol is equally used in pretreatment Object, and process activity yield is relatively low, only 7.4%, it is unsuitable for large-scale production.

Application No. is 200610044594.1 Chinese patent applications to disclose one kind extraction list from spearhead viper venom The method of one ingredient Batroxobin.It discloses spearhead viper venoms through dissolving, staying overnight, centrifuge, Benzamidine Sepharose 6B affinity chromatographys, Sephadex G-25 chromatographies, dialysis, (SP Sepharose, S Sepharose or CM Sepharose) sun Ion column chromatography and (Sephacry1S200, Sephacry1S100 or Superdex-75) sieve chromatography purify to obtain single Component Batroxobin.But the patent chromatographic step is cumbersome, wherein cation seperation column is eluted using step gradient or linear gradient, It is unfavorable for heat source in large-scale production to remove with impurity.

A kind of white-browed snake venom blood coagulation enzyme is disclosed application No. is the Chinese patent application of CN200710099163.X and its is carried Take method and application.It is that Agkistrodon halys ussurriensisEmelianov snake venom is subjected to DEAE-Sephadex A-50 successively it discloses the hemagglutinase Ion-exchange chromatography, Sephadex G-15 gel permeation chromatographies, DEAE-Sephadex A-50 ion-exchange chromatographies and The albumen with blood coagulation activity collected after Sephadex G-75 gel permeation chromatographies.But the patent application uses 4 steps chromatography Method, and chromatography process is mostly linear gradient elution or step gradient, complex technical process.

Application No. is 200510085173.9 Chinese patent applications to disclose a kind of preparation side of Agkistrodon acutus hemocoagulase atrox Method and purposes.It discloses including dissolving snake venom, low-temperature centrifugation, dialysis, DEAE-Sepharose FF chromatographies, dialysis, DEAE- SepharoseFF is chromatographed and Sephadex G25 chromatographic steps.Although the hemagglutinase purifying chromatographic step disclosed in this method is 3 Step, but gradient elution is used, yield on the basis of ensureing purity in purification process is relatively low or heat source removal effect is bad.

In conclusion the extracting method of the snake venom blood coagulation enzyme disclosed in document in the prior art or patent application is difficult to reach It prepares and requires to ideal separation, be not easy to the production of scale.

Invention content

Based on this, the object of the present invention is to provide a kind of methods for extracting hemagglutinase from spearhead viper venom, and the present invention is also Provide the purposes of the hemagglutinase of the present invention.The method and step of extraction hemagglutinase provided by the invention is few, and the period is short, to reduce Process environment exposure；Also, method removal impurity ability provided by the invention is strong, and high income can obtain yield 35% or more, the spearhead haemocoagulase of 98% or more purity.

The purpose of the present invention is what is realized by method comprising the following steps.

On the one hand, the present invention provides a kind of hemagglutinase, the hemagglutinase derives from spearhead viper venom, has following spy Sign：

1) blood clotting enzyme molecular weight is 31731Da, and molecular weight is 25575Da after desaccharification, and N- sugared contents are 19.4%, 3 N- Glycosylation site is respectively N₉₈、N₁₄₆With N₂₂₅；

2) isoelectric point is 5-6；

3) hemagglutinase N-terminal sequence is：VIGGDECDINEHPFL；

4) hemagglutinase includes 6 pairs of disulfide bond, no free sulfhydryl group；

5) hemagglutinase protein sequence contains 232 amino acid.

Preferably, the amino acid sequence of the hemagglutinase such as SEQ ID NO:Shown in 1：

VIGGDECDINEHPFLAFMYYSPQYFCGMTLINQEWVLTAAHCDKTYMRIYLGIHTRSVANDDEVIRYPKEKFICPNK KKNVITDKDIMLIRLNRPVKNSTHIAPISLPSNPPSVGSVCRIMGWGAITTSEDTYPDVPHCANINLFNNTVCREAY NGLPAKTLCAGVLQGGIDTCGGDSGGPLICNGQFQGILSWGSDPCAEPRKPAFYTKVFDYLPWIQSIIAGNKTATCP P。

On the other hand, the present invention provides it is a kind of from spearhead viper venom extract hemagglutinase method, the method includes Following steps：

After spearhead viper venom is pre-processed, pass through diethyllaminoethyl sephadex (DEAE successively Sephadex A-25) ion-exchange chromatography；Benzamidine sepharose gel 4FF (HS) (Benzamidine Sepharose 4FF (HS)) affinity chromatography；And sephadex (Sephadex G-75) gel permeation chromatography, obtain spearhead viper venom Hemagglutinase stoste.

Preferably, it the described method comprises the following steps：

1) spearhead viper venom is dissolved in pH7.0-7.5, the Tris-HCl buffer solutions of 0.01M-0.05M obtain a concentration of 50-200mg/ml, it is therefore preferable to which 80-150mg/ml, the more preferably solution of 100mg/ml are then centrifuged for taking supernatant；

Preferably, the pH value of the Tris-HCl buffer solutions is 7.4, a concentration of 0.02M；

Preferably, the condition of the centrifugation is：At 4 DEG C, 3000rpm-5000rpm, centrifugation time 5-20min, preferably Ground is 10min；

2) supernatant obtained step 1) carries out DEAE Sephadex A-25 ion-exchange chromatographies, using containing 0.2M-0.4M NaCl's, the Tris-HCl buffer solution straight lines elution of pH 7.0-7.5, a concentration of 0.01M-0.05M, according to Absorption peak under 280nm wavelength is collected, and the eluent for including blood coagulation activity component is obtained；

Preferably, the Tris-HCl pH of buffer is 7.4, a concentration of 0.02M；

Preferably, a concentration of 0.3M of NaCl contained by the Tris-HCl buffer solutions；

3) it is with containing 0.5M-1.5M NaCl, pH by the eluent comprising blood coagulation activity component that step 2) obtains The Tris-HCl buffer solutions of 7.0-7.5, a concentration of 0.01-0.06M are dialysed, then carry out Benzamidine Sepharose 4FF (HS) affinity chromatography, using arginic containing 0.05-0.3M, 0.5M-1.5M NaCl's, pH 7.0-7.5 are a concentration of The Tris-HCl buffer solutions of 0.01-0.06M elute, and collect target components；Preferably, the Tris-HCl pH of buffer is 7.0 a concentration of 0.05M；

Preferably, the arginic a concentration of 0.1M contained by the Tris-HCl buffer solutions；

Preferably, a concentration of 1.0M of NaCl contained by the Tris-HCl buffer solutions；

4) target components for collecting step 3) are dense using pH 7.0-7.5 through Sephadex G-75 gel permeation chromatographies After the phosphate buffer elution that degree is 0.05M-0.3M, eluent is collected, spearhead viper venom hemagglutinase stoste is obtained；

Preferably, the pH of the phosphate buffer is 7.4, a concentration of 0.1M；

Preferably, in step 2), before carrying out DEAE Sephadex A-25 ion-exchange chromatographies, pH7.0- is used The Tris-HCl buffer solutions of 7.5, a concentration of 0.01M-0.05M balance 3 column volumes；Preferably, column flow rate 0.8-3ml/ Min, more preferably 2ml/min；Preferably, the pH of the Tris-HCl buffer solutions is 7.4, a concentration of 0.02M.

Preferably, in step 2), the eluent comprising blood coagulation activity component is lived using Quality Control blood plasma coagulo meter Property detection method (as described in patent CN103305591A) and SDS-PAGE (Chinese Pharmacopoeia four general rules of version in 2015 0541,36000 ± 5000Da of molecular weight of albumen) electrophoresis detection, to determine target components.

Preferably, in step 3), before carrying out Benzamidine Sepharose 4FF (HS) affinity chromatography, make It is dialysed, will be contained with the Tris-HCl buffer solutions containing 0.5M-1.5M NaCl, pH 7.0-7.5, a concentration of 0.01-0.06M The eluent of blood coagulation activity component passes through ultrafiltration membrane sterile dialysis 2-3 times；It is highly preferred that the pH of the Tris-HCl buffer solutions is 7.0, a concentration of 0.05M；It is further preferred that a concentration of 1.0M of NaCl contained by the Tris-HCl buffer solutions；Preferably, institute It is ultrafiltration membrane packet to state ultrafiltration membrane；It is highly preferred that the molecular cut off of the ultrafiltration membrane is 10K, it is further preferred that the ultrafiltration Film is polyether sulfone material；

Preferably, in step 3), using Benzamidine Sepharose 4FF (HS) affinity column it Before, using 0.5M-1.5M NaCl are contained, the Tris-HCl buffer solutions of pH 7.0-7.5, a concentration of 0.01-0.06M balance 3 A column volume；Preferably, column flow rate 1.0ml-3ml/min, more preferably 2ml/min；Preferably, the Tris-HCl is slow The pH of fliud flushing is 7.0, a concentration of 0.05M；Preferably, a concentration of 1.0M of NaCl contained by the Tris-HCl buffer solutions；.

Preferably, in step 4), before carrying out Sephadex G-75 gel permeation chromatographies, using pH 7.0-7.5, The phosphate buffer of a concentration of 0.05M-0.3M balances 3 column volumes；Preferably, the flow velocity of column is 0.8-3ml/min, more excellent Selection of land is 2ml/min；Preferably, the pH of the phosphate buffer is 7.4, a concentration of 0.1M.

Preferably, in step 4), after chromatography, eluent is concentrated using the bag filter that molecular weight is 10k that shuts off；It is preferred that Ground, using polyethylene glycol as reverse osmosis dose.

Preferably, by obtained spearhead viper venom hemagglutinase stoste after filtering, packing, freeze-drying, spearhead is made Viper venom hemagglutinase freeze-dried powder.

Preferably, by the spearhead viper venom hemagglutinase freeze-dried powder together with excipient, protective agent, injection lance is made Head viper venom blood clotting enzyme preparation.

In another aspect, being prepared the present invention provides spearhead viper venom hemagglutinase of the present invention and according to the above method Application of the spearhead viper venom hemagglutinase in preparing drug for treating hemorrhagic conditions；

Preferably, the bleeding includes clinical bleeding, operative hemorrhage and various Medicine and Surgery acute bleedings.

Yet another aspect, the present invention provides a kind of pharmaceutical composition for treating bleeding, described pharmaceutical composition packets Include spearhead viper venom hemagglutinase of the present invention and/or the spearhead viper venom hemagglutinase that is prepared according to the above method and Pharmaceutically acceptable carrier.

Compared with prior art, the present invention has the following advantages：

1) step of preparation process of the invention is few, and the period is short, reduces process environment exposure；

2) product yield is high, and technique removes that impurity ability is strong, can obtain 35% or more yield, 98% or more purity lance Head haemocoagulase；

3) product is higher than work, and the protein content that per unit sample contains is less, avoids causing immunogenic response；

4) technology stability is high, and product quality consistency is good, is conducive to clinical application safety.

Description of the drawings

Hereinafter, carry out the embodiment that the present invention will be described in detail in conjunction with attached drawing, wherein：

Fig. 1 is the molecular weight mass spectrogram that hemagglutinase is measured using MALDI-TOF mass spectrums；

The mass spectrum of molecular weight after Fig. 2 is after being sweetened off using ESI-Q-TOF mass spectroscopy hemagglutinasees and desaccharification and NEM alkylations Figure；

Fig. 3 is the RP-HPLC purity using the spearhead haemocoagulase of the method extraction of the present invention；

Fig. 4 is the SDS-PAGE purity using the spearhead haemocoagulase of the method extraction of the present invention；

Fig. 5 is the SDS-PAGE purity using the Agkistrodon halys ussurriensisEmelianov hemagglutinase of the method extraction of the present invention；

Fig. 6 is the SDS-PAGE purity that spearhead haemocoagulase is extracted using existing method；

Fig. 7 is the fiber protein yarn after the fibrin that spearhead viper venom hemagglutinase is formed and use Plasmin digestion；

Fig. 8 is the fiber protein yarn after the fibrin that human thrombin is formed and use Plasmin digestion.

Specific implementation mode

The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.

The hemagglutinase of 98% or more purity can be made in method according to the present invention, then use MALDI-TOF matter It is 31731Da, ESI-Q-TOF mass spectrums (Bu Lu that spectrum (Brooker company micrOTOF-Q II mass spectrographs), which measures blood clotting enzyme molecular weight, Gram company's micrOTOF-Q II mass spectrographs) measure desaccharification after molecular weight be 25575Da, N- sugared contents are 19.4%, including 6 pairs Disulfide bond, no free sulfhydryl group；With 3 N- glycosylation sites, respectively N₉₈、N₁₄₆With N₂₂₅；The voltolisation such as retentive force (IPG) It is 5-6 that burnt method (Bio-Rad handbooks), which measures isoelectric point,；15 amino acid are before Edman sequenators measure hemagglutinase N-terminal： VIGGDECDINEHPFL；

Protein sequence contains 232 amino acid, such as SEQ ID NO:Shown in sequence shown in 1：

SEQ ID NO:1：

Also, Fig. 1 and Fig. 2 are referred to, wherein Fig. 1 is the molecular weight mass spectrogram that hemagglutinase is measured using MALDI-TOF mass spectrums； The mass spectrogram of molecular weight after Fig. 2 is after being sweetened off using ESI-Q-TOF mass spectroscopy hemagglutinasees and desaccharification and NEM alkylations.

The hemagglutinase that the present invention also uses pharmacodynamics test to demonstrate the present invention has anastalsis.

Embodiment 1 extracts hemagglutinase from spearhead viper venom

Extraction step

1. snake venom pre-processes

It is accurate to weigh spearhead viper venom (being purchased from the long-range Nuo Kang Biology Pharmacy Co., Ltd in Liaoning) 80g, it is dissolved in PH7.4, a concentration of 0.02M Tris-HCl buffer solutions obtain a concentration of 100mg/ml solution, are centrifuged at 4 DEG C with 4000rpm 10min takes supernatant.

2.DEAE Sephadex A-25 ion-exchange chromatographies (column I is purchased from GE companies)

PH7.4,0.02M Tris-HCl bufferings are used before chromatographic column (4.5 × 50cm, the magnificent glass chromatography column of Shanghai brocade) loading Liquid balances 3 column volumes, flow velocity：2ml/min.Check that liquid bacterial endotoxin under column, bacterial endotoxin are no more than 0.25EU/ml It is ready for loading.

The supernatant loading in step 1 is taken, with pH7.4, after 0.02M Tris-HCl buffer solutions elute 2 column volumes, is used The above-mentioned Tris-HCl buffer solutions of pH7.4,0.4M NaCl carry out straight line elution, flow velocity about 3ml/min, according under 280nm wavelength Absorption peak collect albumen.

3.Benzamidine Sepharose 4FF (HS) affinity column chromatography (column II is purchased from GE companies)

The elution fraction of collection is through Quality Control blood plasma coagulo meter method determination of activity (patent CN103305591A) and SDS- After the identification of PAGE (four general rules 0541 of Chinese Pharmacopoeia version in 2015) electrophoresis, collect 100 unit of blood clotting enzymatic activity/ml or more and The target components of 36000 ± 5000Da of molecular weight of albumen.Using pH7.0, the 0.05M Tris-HCl buffer solutions of 1.0M NaCl pass through Ultrafiltration membrane packet (Sai Duolisi, polyether sulfone material, molecular cut off 10K) sterile dialysis 3 times.Chromatographic column (1.6 × 30cm, Shanghai Bright and beautiful China's glass chromatography column) before loading with after above-mentioned buffer solution 3 column volumes of balance, by the target components being collected into 2ml/min Flow velocity loading.With above-mentioned buffer solution elute 2 column volumes after, with pH7.0, arginine containing 0.1M, 1.0M NaCl 0.05M Tris-HCl eluents are eluted, flow velocity 2ml/min.Protein peak is collected according to the absorption value under 280nm wavelength, then Desired active ingredients are determined through the coagulo meter method determination of activity of Quality Control blood plasma and SDS-PAGE electrophoresis.

4.Sephadex G-75 column chromatographies (column III is purchased from GE companies)

Before chromatographic column (4.5 × 100cm, the magnificent glass chromatography column of Shanghai brocade) loading, pH 7.4,0.1M phosphate-buffereds are first used Liquid balances 3 column volumes, flow velocity：2ml/min.Check that liquid bacterial endotoxin is ready for loading no more than 0.25EU/ml under column. Affinity chromatography active part is taken, the bag filter for being 10k with the molecular weight that shuts off concentrates, and reverse osmosis dose is polyethylene glycol.It is used after loading Above-mentioned buffer solution elution, elution speed：2ml/min detects efflux with UV detector, collects the protein peak group containing hemagglutinase Point.

Interpretation of result

1. purity detecting

It is examined using electrophoresis system and reversed-phase high performance liquid chromatography (Shimadzu high performance liquid chromatograph 20A, C4 chromatographic column) It surveys, SDS-PAGE is denaturalized non-reduced electrophoresis detection target protein component and shows that a band (as shown in Figure 4), RP-HPLC detections are in Single symmetrical peak (as shown in Figure 3), calculates according to area normalization method, and hemagglutinase content is 99.8%.

2. target protein Activity determination is detected through Quality Control blood plasma coagulo meter method, hemagglutinase in solution after the dissolving of 80g snake venom Gross activity is 5,760,000 units, and the blood clotting enzymatic activity collected after chromatographic purifying is 2,200,000 units, yield 38.2%.

3. target components protein content detects

Hemagglutinase is detected through Forint phenol method (2015 editions Chinese Pharmacopoeias, four general rules, 0731 determining the protein quantity method), and albumen contains Amount is 475mg, than living for 4632 units/mg.

4. prepared by stoste

The protein component of the purity qualification of collection water for injection is dialysed 3 times, 0.22 μm of miillpore filter aseptic filtration, nothing Bacterium dispenses, to get spearhead haemocoagulase freeze-dried powder after freeze-drying.

5. the structure feature of spearhead haemocoagulase

It is 31731Da that A.MALDI-TOF mass spectrums, which measure blood clotting enzyme molecular weight, and ESI-Q-TOF mass spectrums measure molecule after desaccharification Amount is 25575Da, and N- sugared contents are 19.4%, including 6 pairs of disulfide bond, no free sulfhydryl group；With 3 N- glycosylation sites, divide It Wei not N₉₈、N₁₄₆With N₂₂₅；

B. it is 5-6 that retentive force (IPG) isoelectric focussing (Bio-Rad handbooks), which measures isoelectric point,；

15 amino acid are before C.Edman sequenators measure hemagglutinase N-terminal：VIGGDECDINEHPFL；

D. hemagglutinase protein sequence contains 232 amino acid

Embodiment 2 extracts hemagglutinase from spearhead viper venom

Extraction step

1. snake venom pre-processes

Spearhead viper venom 80g accurately is weighed, is dissolved in pH7.0, in 0.04M Tris-HCl buffer solutions, obtains concentration For the solution of 200mg/ml, 20min is centrifuged at 4 DEG C with 3000rpm, takes supernatant.

2.DEAE Sephadex A-25 ion-exchange chromatographies (column I)

PH7.0,0.04M Tris-HCl bufferings are used before chromatographic column (4.5 × 50cm, the magnificent glass chromatography column of Shanghai brocade) loading Liquid balances 3 column volumes, flow velocity：2ml/min.Check that liquid bacterial endotoxin under column, bacterial endotoxin are no more than 0.25EU/ml It is ready for loading.

The supernatant loading in step 1 is taken, with pH7.0, a concentration of 0.01M Tris-HCl buffer solutions elute 2 column volumes Afterwards, with pH7.0, the above-mentioned Tris-HCl buffer solutions progress straight line elution of 0.2M NaCl, flow velocity 3ml/min, according to 280nm waves Absorption peak under long collects albumen.

3.Benzamidine Sepharose 4FF (HS) affinity column chromatography (column II)

The elution fraction of collection is through Quality Control blood plasma coagulo meter method determination of activity (patent CN103305591A) and SDS- After the identification of PAGE (four general rules 0541 of Chinese Pharmacopoeia version in 2015) electrophoresis, collect 100 unit of blood clotting enzymatic activity/ml or more and The target components of 36000 ± 5000Da of molecular weight of albumen.Using containing pH7.0, the 0.05M Tris-HCl buffer solutions of 1.5M NaCl Through ultrafiltration membrane packet (Sai Duolisi, polyether sulfone material, molecular cut off 10K) sterile dialysis 3 times.Chromatographic column (1.6 × 30cm, on Hai Jinhua glass chromatography columns) balance 3 column volumes with above-mentioned buffer solution before loading after, by the target components being collected into 2ml/ Min flow velocity loadings.After 2 column volumes being eluted with above-mentioned buffer solution, with pH7.0, arginine containing 0.05M, 1.5M NaCl 0.05M Tris-HCl eluents are eluted, flow velocity 2ml/min.Protein peak is carried out according to the absorption value under 280nm wavelength It collects, then desired active ingredients is determined through the coagulo meter method determination of activity of Quality Control blood plasma and SDS-PAGE electrophoresis.

4.Sephadex G-75 column chromatographies (column III)

Before chromatographic column (4.5 × 100cm, the magnificent glass chromatography column of Shanghai brocade) loading, pH 7.4, the phosphate of 0.05M are first used Buffer solution balances 3 column volumes, flow velocity：2ml/min.Check that the thin bacterial endotoxin of liquid can be accurate no more than 0.25EU/ml under column Standby loading.Affinity chromatography active part is taken, the bag filter for being 10k with the molecular weight that shuts off concentrates, and reverse osmosis dose is polyethylene glycol.With Above-mentioned buffer solution elution, elution speed：2ml/min.Efflux is detected with UV detector, collects the protein peak group containing hemagglutinase Point.

Interpretation of result

1. purity detecting

It is examined using electrophoresis system and reversed-phase high performance liquid chromatography (Shimadzu high performance liquid chromatograph 20A, C4 chromatographic column) It surveys.SDS-PAGE is denaturalized non-reduced electrophoresis detection target protein component and shows that a band, RP-HPLC detections are in single symmetrical peak, It is calculated according to area normalization method, hemagglutinase content is 98.2%.

2. target protein Activity determination

It being detected through Quality Control blood plasma coagulo meter method, hemagglutinase gross activity is 5,820,000 units in solution after the dissolving of 80g snake venom, The blood clotting enzymatic activity collected after chromatographic purifying is 2,050,000 units, yield 35.2%.

3. target components protein content detects

Hemagglutinase is detected through Forint phenol method (2015 editions Chinese Pharmacopoeias, four general rules, 0731 determining the protein quantity method), and albumen contains Amount is 502mg, than living for 4084 units/mg.

The coagulation function of 3 hemagglutinase of embodiment is studied

This study tour spearhead haemocoagulase is in vitro experiment to hemorrhagic disease patients Wits and to fibre The influence that fibrillarin is formed

Experiment one

1. experiment purpose

Spearhead haemocoagulase is investigated to hemorrhagic disease patients blood plasma activated partial thromboplastin time (APTT), blood coagulation The influence of zymogen time (PT), factor X activation (F Ⅹ) and the generation of fibrin ferment.

2. experimental subjects and method

Sample prepares and reagent：According to Principles in Informed Consent, the 3.8% sodium citrate anticoagulation of 20 normal persons is left and taken Slurry, age 20-45 Sui, men and women is fifty-fifty.Hemophilia A patients 25, vitamin K-dependent clotting factor deficiency disease patient 7, blood Pipe hemophilia (Von Willebrand ' s disease, VWD) patient 3, Ⅹ deficiency disease patients of F 3, hemophilia B, F V Deficiency disease, VII deficiency diseases of F and each 1 of afibrinogenemia patient.It is saved backup at -20 DEG C after blood plasma packing.

Test medicine：It is prepared by spearhead haemocoagulase, method described according to embodiments of the present invention 1；

Human plasma factor X (F Ⅹ), the production of Calbiochem companies；

Chromophoric substrate S-2337, S-2238, the production of Sigma companies；

Activate (FXa) standard items of F Ⅹ and human thrombin standard items, the production of Hyphen BioMed companies.

Instrument：STAGO COMPACT Automatic coagulometers (French Stago companies)；All-wave length multi-function microplate reader (Sai Mo Flying generation, you are scientific and technological)；KDC-40 low speed centrifuges (Yi Cheng laboratory equipments Co., Ltd of Southeast Region of Beijing)

3. experimental method

3.1 activated partial thromboplastin times (APTT) are detected with prothrombin time (PT)

It is separately added into certain density spearhead haemocoagulase in human normal plasma and hemorrhagic disease patients blood plasma, 37 APTT, PT are detected on Automatic coagulometer after DEG C incubating 3min, and compared with blank control group (be added normal saline). (APTT reference ranges：28s-40s, PT reference range：11s-14.5s.)

3.2 Chromogenic assays detect the activation of plasma F Ⅹ and the generation of fibrin ferment

S-2337 and S-2238 is Ⅹ a of F and Thrombin specificity chromophoric substrate respectively, and chromophore is released after hydrolyzed The burst size of paranitroanilinum, the latter is directly proportional to Ⅹ a of F and the vigor of fibrin ferment, and absorption peak is detected at 405nm.

Blood plasma or F Ⅹ (8 μ g/ml) 100 μ l are taken, 100 μ l spearhead haemocoagulase solution [spearhead haemocoagulases are added 0.02U/ml, Tris-HCl 100mmol/L (PH 7.5), CaCl₂6mmol/L, hydrochloric acid methyl phenyl ethers anisole 2mmol/L], 37 DEG C of incubations 5min, draw 100 μ l be added in 96 hole elisa Plates, add 100 μ l chromophoric substrates buffer solutions [S-2337 0.625mg/ml, Tris-HCl 0.5mmol/L (PH 8.3), EDTA 25mmol/L, NaCl 375mmol/L], its extinction is detected at 405nm Angle value 30min.

100 μ l of blood plasma are taken, 1 μ l of F Ⅹ A (0.1U/ml) solution are added, after 37 DEG C incubate 5min, draw 10 μ l points in 96 holes In ELISA Plate, 96 μ l chromophoric substrates buffer solutions (S-2238 2mmol/L, EDTA 5mmol/L, 50 μ of benzamidine hcl are added Mol/L, CaCl₂3mmol/L), its absorbance value 30min is detected at 405nm.

4. experimental result：

Spearhead haemocoagulase can shorten the APTT of human normal plasma, in a concentration of 0.05U/ml, can make normal person's blood The APTT of slurry shortens 10s or so；Spearhead haemocoagulase energy dose dependent shortens haemophiliac plasma A PTT, a concentration of When 0.05U/ml, it is horizontal (as shown in table 1) that normal person can be reached.Spearhead haemocoagulase is to normal person and blood coagulation disorders disease People PT has no significant effect (as shown in table 2).

Influence of the 1 spearhead haemocoagulase of table to normal person and haemophiliac APTT

*P<0.05, * * * P<0.001vs haemophiliac (n=25)；###P<0.001vs normal person (n=6)

Influence of the 2 spearhead haemocoagulase of table to normal person and blood coagulation disorders patient PT

Spearhead haemocoagulase generates without notable shadow normal person and blood coagulation disorders patients blood plasma FX activation and fibrin ferment It rings.

5. conclusion

Spearhead haemocoagulase can significantly shorten normal person, the blood coagulation disorders human plasma APTT times, to PT, FX activation, Fibrin ferment generation does not make significant difference.

Experiment two

1. experiment purpose

Application scanning Electronic Speculum investigates the influence that spearhead haemocoagulase forms fibrin.

2. experiment material and method

According to Principles in Informed Consent, the 3.8% sodium citrate anti-freezing blood plasma of 20 normal persons, age 20-45 Sui, men and women are taken It is fifty-fifty.

Test medicine：It is prepared by spearhead haemocoagulase, method described according to embodiments of the present invention 1；Human thrombin, purchase From sigma；

Scanning electron microscope：CamScan 3400；

Scanning electron microscope experimental method：500 μ l of human normal plasma are taken to be put into 4 1.5ml centrifuge tubes and number respectively.In 1- People's blood coagulation that 500U/ml is added in 5 μ l, the 3-4 centrifuge tubes of spearhead haemocoagulase of 5U/ml is separately added into No. 2 centrifuge tubes 5 μ l of enzyme.Each centrifuge tube is put to 37 DEG C of water-bath 30min, then the fibrinolysin (rt- of 5mg/ml is added into 2, No. 4 centrifuge tubes respectively PA) 5 μ l are gently mixed water-bath 15min after mixing.Gently choose the fibrin clot in each centrifuge tube, physiological saline is used in combination After cleaning 3 times, fixes rear inspection with 2.5% glutaraldehyde solution and do scanning electron microscope (as shown in Figure 7 and Figure 8 respectively).Through computer point It is counted after analysing the width of fiber protein yarn.

3. experimental result

Electronic Speculum the results show that spearhead haemocoagulase can promote to be formed relatively fine fiber protein yarn (153.5 ± 12.4μm).The fiber protein yarn that hemagglutinase is formed passes through the digestion of fibrinolysin, thickens, swelling；The fibre formed with fibrin ferment Fibrillarin silk is relatively easier to be digested.This shows spearhead haemocoagulase and human thrombin difference, promotes in bleeding part Fibrin formation is very unstable, easily by plasmin degradation, therefore does not easily lead to the generation of thrombus complication.

4. conclusion

Spearhead haemocoagulase can promote fibrinogen to form digestible fibrin.Compared with fibrin ferment, no Easily form thrombus.

Comparative example 1 extracts hemagglutinase from Agkistrodon halys ussurriensisEmelianov snake venom

Extraction step

1. snake venom pre-processes

Agkistrodon halys ussuriensis snake venom 80g accurately is weighed, is dissolved in pH7.4, a concentration of 0.02M Tris-HCl buffer solutions obtain A concentration of 100mg/ml solution centrifuges 10min at 4 DEG C with 4000rpm, takes supernatant.

2.DEAE Sephadex A-25 ion-exchange chromatographies (column I)

The supernatant loading in step 1 is taken, with pH7.4, after 0.02M Tris-HCl buffer solutions elute 2 column volumes, is used The above-mentioned Tris-HCl buffer solutions of pH7.4,0.4M NaCl carry out straight line elution, flow velocity 3ml/min, according under 280nm wavelength Absorption peak is collected.

3.Benzamidine Sepharose 4FF (HS) affinity column chromatography (column II)

The elution fraction of collection is through Quality Control blood plasma coagulo meter method determination of activity (patent CN103305591A) and SDS- After the identification of PAGE (four general rules 0541 of Chinese Pharmacopoeia version in 2015) electrophoresis, collect 100 unit of blood clotting enzymatic activity/ml or more and The target components of molecular weight of albumen 36000-43000Da.Using containing pH7.0, the 0.05M Tris-HCl buffer solutions of 1.0M NaCl Through ultrafiltration membrane packet (Sai Duolisi, polyether sulfone material, molecular cut off 10K) sterile dialysis 3 times.Chromatographic column (1.6 × 30cm, on Hai Jinhua glass chromatography columns) balance 3 column volumes with above-mentioned buffer solution before loading after, by the target components being collected into 2ml/ Min flow velocity loadings.With above-mentioned buffer solution elute 2 column volumes after, with pH7.0, arginine containing 0.1M, 1.0M NaCl 0.05M Tris-HCl eluents are eluted, flow velocity 2ml/min.Protein peak is collected according to the absorption value under 280nm wavelength, then Desired active ingredients are determined through the coagulo meter method determination of activity of Quality Control blood plasma and SDS-PAGE electrophoresis.

4.Sephadex G-75 column chromatographies (column III)

Before chromatographic column (4.5 × 100cm, the magnificent glass chromatography column of Shanghai brocade) loading, pH 7.4,0.1M phosphate-buffereds are first used Liquid balances 3 column volumes, flow velocity：2ml/min.Check that the thin bacterial endotoxin of liquid is ready for no more than 0.25EU/ml under column Sample.Affinity chromatography active part is taken, the bag filter for being 10k with the molecular weight that shuts off concentrates, and reverse osmosis dose is polyethylene glycol.With above-mentioned Buffer solution elutes, elution speed：2ml/min.Efflux is detected with UV detector, collects the protein peak component containing hemagglutinase.

Interpretation of result

1. purity detecting

It is examined using electrophoresis system and reversed-phase high performance liquid chromatography (Shimadzu high performance liquid chromatograph 20A, C4 chromatographic column) It surveys.SDS-PAGE is denaturalized non-reduced electrophoresis detection target protein component and shows 2 bands (as shown in Figure 5), and RP-HPLC detections are in more A peak, calculates according to area normalization method, and hemagglutinase content is only 93.7%.

2. target protein Activity determination

It is detected through Quality Control blood plasma coagulo meter method, hemagglutinase gross activity is 125 in solution after the dissolving of 80g agkistrodon halys ussuriensis snake venom Ten thousand units, the blood clotting enzymatic activity collected after chromatographic purifying are 19.25 ten thousand units, yield 15.4%.

3. target components protein content detects

Hemagglutinase is detected through Forint phenol method (2015 editions Chinese Pharmacopoeias, four general rules, 0731 determining the protein quantity method), and albumen contains Amount is 178mg, than living for 1887 units/mg.

4. concrete outcome is relatively shown in Table 3.

3 the method for the present invention of table extracts agkistrodon halys ussuriensis and spearhead haemocoagulase result

5. conclusion

Hemagglutinase is extracted from Agkistrodon halys ussurriensisEmelianov snake venom with the method for the present invention, hemagglutinase yield is only 15.4%, Purity is 93.7%, is 1081 units/mg than living, and various indexs are below spearhead viper venom hemagglutinase.This patent method is special It is not suitable for the extraction of spearhead viper venom hemagglutinase, meanwhile, it is applicable in the spearhead viper venom blood clotting of the method extraction of the present invention Enzyme content is higher, is more suitable for the production of scale.

2 existing method of comparative example extracts hemagglutinase from spearhead viper venom

Extraction step(application reference number is the Chinese patent of CN200710099163.X)

1. snake venom pre-processes

Spearhead viper venom 10g accurately is weighed, with pH7.5,0.05M Tris-HCl buffer solutions are that 250mg/ml is molten Liquid centrifuges 10min with 3000rpm, takes supernatant.

2.DEAE Sephadex A-50 ion-exchange chromatographies (column I)

PH7.5,0.05M Tris-HCl buffer solutions are used before chromatographic column (5 × 100cm, the magnificent glass chromatography column of Shanghai brocade) loading Balance 3 column volumes, flow velocity：1ml/min.The supernatant loading in step 1 is taken, after eluting 2 column volumes with above-mentioned buffer solution, With the Tris- of the pH7.5 of the 0.75M containing NaCl of 1000ml pH7.5, the Tris-HCl buffer solutions of 0.05M and equivalent, 0.05M HCl buffer solutions carry out linear gradient elution, flow velocity 1ml/min.Protein peak is received according to the absorption value under 280nm wavelength Collection.

3.Sephadex G-15 chromatography desalinations (column II)

Chromatographic column (column 5 × 100cm of specification, the magnificent glass chromatography column of Shanghai brocade), the elution fraction of collection is through Quality Control blood plasma blood Solidifying instrument method determination of activity (patent CN103305591A) and SDS-PAGE (four general rules 0541 of Chinese Pharmacopoeia version in 2015) electricity After the identification of swimming method, the target components of 100 unit of blood clotting enzymatic activity/36000 ± 5000Da of ml or more and molecular weight of albumen are collected.Layer After balancing 3 column volumes with water for injection before analysis loading, by the target components being collected into 1ml/min flow velocity loadings.With injection It is eluted with water, same procedure collects the component with blood coagulation activity.

4.DEAE Sephadex A-50 ion exchanges chromatograph (column III) again

The pH to 5.2 for the collection liquid with blood coagulation activity that regulating step 3 is collected, is then chromatographed, chromatographic column (column again 5 × 100cm of specification, the magnificent glass chromatography column of Shanghai brocade) preceding pH 5.2 is chromatographed, the Tris-HCl of 0.05M balances 3 column volumes Afterwards, by the target components being collected into 1ml/min flow velocity loadings, then the Tris-HCl buffer solutions of 1000ml pH5.2,0.05M and The pH5.2 of the 0.25M containing NaCl of equivalent, the Tris-HCl buffer solutions of 0.05M carry out linear gradient elution, flow velocity 1ml/ min；The part with blood coagulation activity in eluent is collected with method identical with step 3.

5.Sephadex G-75 column chromatographies (column IV)

It is existing before chromatography that chromatographic column (column 5 × 100cm of specification, the magnificent glass chromatography column of Shanghai brocade) is balanced 3 with water for injection Column volume, flow velocity 1ml/min；When chromatography, after supernatant is moved fully to glue surface once, water for injection is added above glue surface Then 50ml is eluted with water for injection, flow velocity 1ml/min；Being collected with method identical with step 3 has blood coagulation in eluent Active part.

Interpretation of result

1. purity detecting

It is examined using electrophoresis system and reversed-phase high performance liquid chromatography (Shimadzu high performance liquid chromatograph 20A, C4 chromatographic column) It surveys.SDS-PAGE is denaturalized non-reduced electrophoresis detection target protein component and is shown as 2 bands (as shown in Figure 6), and RP-HPLC detections are in Multiple peaks, calculate according to area normalization method, and hemagglutinase content is only 94.2%.

2. target protein Activity determination

It is detected through Quality Control blood plasma coagulo meter method, hemagglutinase gross activity is 71 in solution after the dissolving of 10g agkistrodon halys ussuriensis snake venom Ten thousand units, the blood clotting enzymatic activity collected after chromatographic purifying are 5.75 ten thousand units, yield 8.1%.

3. target components protein content detects

Hemagglutinase is detected through Forint phenol method (2015 editions Chinese Pharmacopoeias, four general rules, 0731 determining the protein quantity method), and albumen contains Amount is 18mg, than living for 3194 units/mg.

4. concrete outcome is relatively shown in Table 4.

4 existing method of table extracts spearhead haemocoagulase result with the method for the present invention

5. conclusion

Spearhead viper venom hemagglutinase is extracted with existing method, hemagglutinase purity and than work it is fine, respectively 94.2% and 3194 units/mg, but its yield is far below the yield 38.2% obtained by the method for the present invention, only 8.1%.This patent Method purification step is less, only 3 steps, and the production cycle is shorter, yield higher, is particularly suited for hemagglutinase large-scale production.Together When, can ensure high than living, and avoid causing immunogenic response.

Although present invention has been a degree of descriptions, it will be apparent that, do not departing from the spirit and scope of the present invention Under the conditions of, the appropriate variation of each condition can be carried out.It is appreciated that the present invention is not limited to the embodiments, and it is attributed to right It is required that range comprising the equivalent replacement of each factor.

Sequence table

<110>The long-range Nuo Kang Biology Pharmacy Co., Ltd in Liaoning

<120>The hemagglutinase and its preparation method and application extracted from Brazilian spearhead viper venom

<130> DIC16110037

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 232

<212> PRT

<213>Artificial sequence

<220>

<223>The amino acid sequence of hemagglutinase

<400> 1

Val Ile Gly Gly Asp Glu Cys Asp Ile Asn Glu His Pro Phe Leu Ala

1 5 10 15

Phe Met Tyr Tyr Ser Pro Gln Tyr Phe Cys Gly Met Thr Leu Ile Asn

20 25 30

Gln Glu Trp Val Leu Thr Ala Ala His Cys Asp Lys Thr Tyr Met Arg

35 40 45

Ile Tyr Leu Gly Ile His Thr Arg Ser Val Ala Asn Asp Asp Glu Val

50 55 60

Ile Arg Tyr Pro Lys Glu Lys Phe Ile Cys Pro Asn Lys Lys Lys Asn

65 70 75 80

Val Ile Thr Asp Lys Asp Ile Met Leu Ile Arg Leu Asn Arg Pro Val

85 90 95

Lys Asn Ser Thr His Ile Ala Pro Ile Ser Leu Pro Ser Asn Pro Pro

100 105 110

Ser Val Gly Ser Val Cys Arg Ile Met Gly Trp Gly Ala Ile Thr Thr

115 120 125

Ser Glu Asp Thr Tyr Pro Asp Val Pro His Cys Ala Asn Ile Asn Leu

130 135 140

Phe Asn Asn Thr Val Cys Arg Glu Ala Tyr Asn Gly Leu Pro Ala Lys

145 150 155 160

Thr Leu Cys Ala Gly Val Leu Gln Gly Gly Ile Asp Thr Cys Gly Gly

165 170 175

Asp Ser Gly Gly Pro Leu Ile Cys Asn Gly Gln Phe Gln Gly Ile Leu

180 185 190

Ser Trp Gly Ser Asp Pro Cys Ala Glu Pro Arg Lys Pro Ala Phe Tyr

195 200 205

Thr Lys Val Phe Asp Tyr Leu Pro Trp Ile Gln Ser Ile Ile Ala Gly

210 215 220

Asn Lys Thr Ala Thr Cys Pro Pro

225 230

Claims

1. a kind of hemagglutinase, the hemagglutinase derives from spearhead viper venom, has following characteristics：

1) blood clotting enzyme molecular weight is 31731Da, and molecular weight is 25575Da after desaccharification, and N- sugared contents are 19.4%, 3 N- glycosyls It is respectively N to change site₉₈、N₁₄₆With N₂₂₅；

2) isoelectric point is 5-6；

3) hemagglutinase N-terminal sequence is：VIGGDECDINEHPFL；

5) hemagglutinase protein sequence contains 232 amino acid；

VIGGDECDINEHPFLAFMYYSPQYFCGMTLINQEWVLTAAHCDKTYMRIYLGIHTRSVANDDEVIRYPKEKFI CPNKKKNVITDKDIMLIRLNRPVKNSTHIAPISLPSNPPSVGSVCRIMGWGAITTSEDTYPDVPHCANINLFNNTVC REAYNGLPAKTLCAGVLQGGIDTCGGDSGGPLICNGQFQGILSWGSDPCAEPRKPAFYTKVFDYLPWIQSIIAGNKT ATCPP。

2. a kind of method for extracting hemagglutinase from spearhead viper venom, the described method comprises the following steps：

After spearhead viper venom is pre-processed, pass through diethyllaminoethyl sephadex (DEAE Sephadex successively A-25) ion-exchange chromatography；Benzamidine sepharose gel 4FF (HS) (Benzamidine Sepharose 4FF (HS)) is affine Chromatography；And sephadex (Sephadex G-75) gel permeation chromatography, obtain spearhead viper venom blood clotting proenzyme Liquid.

3. according to the method described in claim 2, it is characterized in that, the described method comprises the following steps：

1) spearhead viper venom is dissolved in the Tris-HCl buffer solutions of pH 7.0-7.5,0.01M-0.05M, obtains a concentration of 50- 200mg/ml, it is therefore preferable to which 80-150mg/ml, the more preferably solution of 100mg/ml are then centrifuged for taking supernatant；

Preferably, the condition of the centrifugation is：At 4 DEG C, 3000rpm-5000rpm, centrifugation time 5-20min, it is therefore preferable to 10min；

2) supernatant obtained step 1) carries out DEAE Sephadex A-25 ion-exchange chromatographies, using containing 0.2M- 0.4M NaCl, pH 7.0-7.5, a concentration of 0.01M-0.05M Tris-HCl buffer solution straight lines elution, according to 280nm waves Absorption peak under long is collected, and the eluent for including blood coagulation activity component is obtained；

Preferably, the pH of the Tris-HCl buffer solutions is 7.4, a concentration of 0.02M；

3) eluent comprising blood coagulation activity component for obtaining step 2) is with containing 0.5M-1.5M NaCl, pH 7.0- 7.5, the Tris-HCl buffer solutions dialysis of a concentration of 0.01-0.06M, then carry out Benzamidine Sepharose 4FF (HS) Affinity chromatography, using containing 0.05-0.3M arginine, 0.5M-1.5M NaCl, pH 7.0-7.5, a concentration of 0.01-0.06M Tris-HCl buffer solutions elution, collect target components；

Preferably, the pH of the Tris-HCl buffer solutions is 7.0, a concentration of 0.05M；

Preferably, arginic a concentration of 0.1M contained by the Tris-HCl buffer solutions；

4) target components for collecting step 3) are a concentration of using pH7.0-7.5 through Sephadex G-75 gel permeation chromatographies After the phosphate buffer elution of 0.05M-0.3M, eluent is collected, spearhead viper venom hemagglutinase stoste is obtained；

Preferably, the pH of the phosphate buffer is 7.4, a concentration of 0.1M.

4. according to the method described in claim 3, it is characterized in that, in step 2), DEAE Sephadex A-25 are being carried out Before ion-exchange chromatography, 3 cylinders are balanced using the Tris-HCl buffer solutions of pH 7.0-7.5, a concentration of 0.01M-0.05M Product；

Preferably, column flow rate 0.8-3ml/min, more preferably 2ml/min；

Preferably, the pH of the Tris-HCl buffer solutions is 7.4, a concentration of 0.02M.

5. method according to claim 3 or 4, which is characterized in that in step 2), using blood plasma coagulo meter to the packet The eluent of the component containing blood coagulation activity carries out Activity determination and uses SDS-PAGE electrophoresis to described comprising blood coagulation activity group The eluent divided is detected, to determine target components.

6. according to the method described in any one of claim 3-5, which is characterized in that in step 3), carrying out Before Benzamidine Sepharose 4FF (HS) affinity chromatography, the NaCl containing 0.5M-1.5M, pH 7.0- are used 7.5, a concentration of 0.01-0.06M Tris-HCl buffer solutions dialysis, by the eluent of the component containing blood coagulation activity by ultrafiltration membrane without Bacterium is dialysed 2-3 times；

Preferably, a concentration of 1.0M of the NaCl contained by the Tris-HCl buffer solutions；

Preferably, the ultrafiltration membrane is ultrafiltration membrane packet；It is highly preferred that the molecular cut off of the ultrafiltration membrane is 10K, it is further excellent Selection of land, the ultrafiltration membrane are polyether sulfone material；

Preferably, it in step 3), before Benzamidine Sepharose 4FF (HS) affinity chromatography, uses NaCl containing 0.5M-1.5M, pH 7.0-7.5, a concentration of 0.01-0.06M Tris-HCl buffer solutions balance 3 cylinders Product；

Preferably, column flow rate 1.0ml-3ml/min, more preferably 2ml/min；

Preferably, a concentration of 1.0M of the NaCl contained by the Tris-HCl buffer solutions.

7. according to the method described in any one of claim 3-6, which is characterized in that in step 4), carrying out Sephadex Before G-75 gel permeation chromatographies, 3 cylinders are balanced using the phosphate buffer of pH 7.0-7.5, a concentration of 0.05M-0.3M Product；

Preferably, the flow velocity of column is 0.8-3ml/min, more preferably 2ml/min；

Preferably, the pH of the phosphate buffer is 7.4, a concentration of 0.1M；

Preferably, in step 4), after chromatography, eluent is concentrated using the bag filter that molecular weight is 10k that shuts off；Preferably, Using polyethylene glycol as reverse osmosis dose.

8. according to the method described in any one of claim 2-7, which is characterized in that the method further includes the spearhead that will be obtained Spearhead viper venom hemagglutinase freeze-dried powder is made after filtering, packing, freeze-drying in viper venom hemagglutinase stoste；

Preferably, by the spearhead viper venom hemagglutinase freeze-dried powder together with excipient, protective agent, injection spearhead Pallas pit viper is made Snake snake venom blood coagulation enzyme preparation.

9. hemagglutinase prepared by the method according to any one of claim 2-8.

10. fibrin ferment prepared by method described in any one of hemagglutinase according to claim 1 or claim 2-8 or Application of the hemagglutinase according to claim 9 in preparing the drug for treating hemorrhagic conditions；